The arsenal of weapons for treating virus infections is relatively meager. Although vaccines for some viruses can be effective, for other viruses, antiviral agents are needed. Among potential targets for antiviral therapy that arise during certain viral infections are the virus-coded proteinases. These enzymes, essential for the synthesis of infectious virus, are required to process virus-specific precursor proteins involved in the maturation, assembly and replication of such pathogenic human viruses as adenovirus. poliovirus. hepatitis C virus, and human immunodeficiency virus. Viruscoded proteinases are highly specific for their substrates such that if equally specific inhibitors can be obtained, they should interfere with virus replication and not normal cellular metabolism. Our model system for the development of new antiviral agents is human adenovirus. One specific aim is to understand at the biochemical and structural levels how the activity of the adenovirus proteinase (AVP) is regulated. Our laboratory has shown that AVP is inactive and requires cofactors that restrict its activity in both space and time. One cofactor is pVIc, an 11 amino acid viral peptide; another is the viral DMA; actin is a cellular cofactor. The cofactors increase the kcat/Km for substrate hydrolysis. We determined the crystal structure of AVP-pVIc to a resolution of 1.6 A and of AVP to 0.98 A. The fold of the protein was unique; AVP represents the first member of a new class of cysteine proteinases. It is activated by pVIc via a 54 amino acid long signal transduction pathway. The other specific aim is to use this information to identify drug targets in AVP. in its cofactors. and in its substrates and to use structure-based drug design to discover compounds that bind to these targets. A novel aspect of the work is in identifying drug targets other than those in the active site. Because of the signal transduction pathway, more than 25% of the surface of AVP is a legitimate drug target. One compound, a pathway blocker, is predicted to bind about 15 Angstroms from the active site; it inhibits activation of AVP by pVIc. A novel class of inhibitors was designed- substrate inhibitors. These bind to a cleavage site on a substrate thereby preventing its recognition by the proteinase. Although the focus of this grant proposal is to obtain antiviral agents against adenovirus, we describe how ideas that arose out of this project can be applied to the development of antiviral agents against SARS Coronavirus and influenza virus.